ABSTRACT Current breast cancer therapy is largely based on targeted therapeutics. In particular, hormonal therapy targets the estrogen receptor (ER) in ER-positive cancers, whereas cancers that overexpress HER2 are treated with different types of HER2-targeted drugs. The latter include monoclonal antibodies, small molecule inhibitors, or HER2-antibodies conjugated to a cytotoxic drug. Other targeted drugs used in breast cancer therapy act on CDK4/6, mTOR, EGFR, HDAC or PARP. Unfortunately, many patients do not respond to targeted therapies or respond initially and then develop resistance. Identification of new ?druggable? mediators of the oncogenic effects of the current breast cancer drug targets could yield clinical improvements in the treatment of cancers resistant to targeted therapy. The common endpoint of essentially all the signal transduction pathways mediated by cancer drug targets is an effect on transcription of genes that regulate tumor growth, survival and metastasis. The most targetable proteins in the transcription space are transcription-regulating kinases belonging to the cyclin-dependent kinase (CDK) family, CDK7, CDK8/19 and CDK9. Inhibitors of these kinases are undergoing preclinical and clinical development. We hypothesize that inhibitors of transcription-regulating kinases will synergize with targeted drugs used to treat breast cancer, and that combinations of targeted drugs with transcription-regulating kinase inhibitors may overcome or prevent the development of resistance to targeted drugs. We have identified several synergistic combinations of targeted breast cancer drugs and transcription-regulating kinase inhibitors, most notably the combinations of CDK8/19 inhibitors with ER- and HER2-targeting drugs and CDK7 inhibitors with EGFR-targeting drugs. In the remaining period of the project, we will characterize the most potent of the discovered combinations, namely a combination of HER2 and CDK8/19 inhibitors. Under Aim 1, the efficacy of a combination between HER2 inhibitor lapatinib and a CDK8/19 inhibitor will be tested in vivo, using xenograft models of lapatinib-sensitive and resistant HER2- positive breast cancer cells. Aim 2 is to investigate the mechanism of the synergy between lapatinib and CDK8/19 inhibitors. The experimental plan for this Aim is based on the results of RNA-Seq analysis that identified candidate downstream mediators of this synergy. Expression of these candidate genes is regulated by transcription factors, phosphorylation of which is dependent on both CDK8/19 and HER2. Aim 3 is to develop a conjugate between an anti-HER2 monoclonal antibody and a potent CDK8/19 inhibitor and to test the in vitro efficacy of this conjugate, relative to the unconjugated agents. The success of this project will pave the way towards improving the efficacy of targeted therapy in breast cancer through novel combinations of the current targeted drugs with transcription-regulating kinase inhibitors. Future basic and translational research on the combinatorial effects of HER2- and CDK8/19-targeted drugs may have a profound impact on the treatment of metastatic HER2-positive breast cancers that are resistant to HER2-targeted drugs.